Arginine mimetics as factor Xa inhibitors

ABSTRACT

The invention relates generally to a novel type of arginine mimetics which are inhibitors of factor X a ; to pharmaceutical compositions which comprise these mimetics; and to the use of these arginine mimetics for producing compositions for antithrombotic therapy.

The invention relates generally to a novel type of arginine mimetics,which are inhibitors of factor X_(a); to pharmaceutical compositionswhich comprise these mimetics; and to the use of these arginine mimeticsfor producing medicines for antithrombotic therapy.

Proteins, such as thrombin, which are involved in the blood coagulationcascade have for many years now been potential targets in the treatmentof vascular diseases, with the aim of inhibiting of them and therebyavoiding thrombotic vascular occlusions or reopening thromboticallyoccluded blood vessels. The use of conventional anticoagulants whichcontain thrombin inhibitors is problematical since they increase theprobability of bleeding complications (G. J. Phillipides and J. Loscalzo(1996), Coronary Artery Dis., 7, 497–507). Furthermore, directlyinhibiting thrombin does not interrupt the production of thrombin fromprothrombin. In this case, therefore, it is necessary to supplyrelatively high doses of inhibitor continuously in order to maintain anantithrombotic effect in vivo.

Consequently, in the search for novel antithrombotic medicines,inhibition of the blood coagulation factor X_(a) became a main targetfor developing active compounds. Factor X_(a) is a trypsin-like serineprotease which converts the zymogen prothrombin into its active formthrombin. By inhibiting factor X_(a), therefore, it is possible toprevent thrombin being formed while a level of thrombin activity whichis required for primary hemostasis is maintained (F. Al-Obeidy and J. A.Ostrem (1998), Drug Discovery Today, 3, 223–231).

A large number of factor X_(a) inhibitors are nowadays known. Thedevelopment of a number of these factor X_(a) inhibitors is based onconserving the structural motif Gly-Arg (with Gly being the P2 radicaland Arg being the P1 radical, i.e. Gly binds in the S2 pocket and Argbinds in the S1 pocket of the factor X_(a) protein) at the site at whichthe prothrombin is cleaved by factor X_(a). In this connection, a largenumber of syntheses, in which a phenylalanine radical which issubstituted on its phenyl ring by a basic amidino group is used as amimetic for the Arg radical, have been described for factor X_(a)inhibitors (J. Stürzebecher et al., (1989), Thromb. Res. 54, 245–252).It has been found that the factor X_(a) inhibitors in this series whichhave thus far been most effective are derivatives of3′-amidinophenylalanine.

Taking the guiding structureN^(α)-tosylglycyl-D,L-3-amidinophenylalanine alkyl ester (Compound 1 inFIG. 1; J. Stürzebecher et al., see above) as a starting point, a largenumber of peptidic bisbenzamidine compounds have been developed. Themost powerful factor X_(a) inhibitor (K_(i)=0.5 μM) from this series isN^(α)-4-amidinobenzenesulfonylglycyl-D,L-4-amidinophenylalanine ethylester (Compound 2 in FIG. 1; B. Gabriel et al., (1998), J. Med. Chem.41, 4240–4250), which binds “inversely” to factor X_(a): its4′-amidinobenzenesulfonyl group lies in the S1 pocket of factor X_(a),while the remainder of the molecule, together with the glycyl spacer,projects into the hydrophobic S3/S4 binding sites, thereby makingpossible additional interactions with the electronegative cavity, whichis formed by the carbonyl oxygens of Lys 96, Thr. 98 and Glu 97 and itscarboxylate group, behind the hydrophobic S3/S4 region.

According to the prior art, the group which is linked N-terminally to anamidinophenylalanine radical or to another arginine mimetic constitutesthe P3/P4 radical of the potential factor X_(a) inhibitor, with theN-terminally linked group preferably being bonded to the argininemimetic by way of a glycine spacer and a sulfonamide group.

Since then, nonpeptide bisbenzamidine compounds which possess markedlyimproved inhibitor properties (K_(i)=34 nM), and which are characterizedby a shorter distance between the two aromatic groups, have beenobtained (T. P. Maduskuie et al., (1998), J. Med. Chem. 41, 53–62). Onthe basis of modeling analyses, it is assumed that an inhibitor 3 ofthis nature (see FIG. 1) extends, by means of the m-benzamidine group,into the S1 pocket, and interacts in this pocket with the Asp 189radical, and, by means of the p-benzamidine group, into the S4aryl-binding pocket, where it enters into cation-π interactions andhydrophobic interactions with the surrounding radicals Phe 174, Tyr 99and Trp 215.

Against this background, an object of the invention is to provide novelinhibitors of factor X_(a) which are highly efficient and highlyspecific.

In addition, an important object of the invention is to point outpossibilities for using the compounds according to the invention forproducing a medicine for antithrombotic therapy.

Other objects and advantages of the invention ensue from the followingdescription.

These objects are achieved by the subject-matter of the independentclaims, in particular based on the provision of the compounds accordingto the invention in accordance with the structural formula I.

Advantageous embodiments are described in the subclaims.

The object is achieved, according to the invention, by providing ahighly efficient and highly selective factor X_(a) inhibitor whichcomprises an arginine mimetic which possesses a N-terminal radical and aC-terminal radical, with the conformation of the inhibitor enablingintermolecular interactions to take place between the C-terminal radicaland the S3/S4 pocket of the factor X_(a) protein. A binding mode of thisnature, in which the C-terminal radical extends into the S3/S4 bindingpocket of the factor X_(a) protein is particularly advantageous since,in the case of the factor X_(a) inhibitor according to the invention,the C-terminal radical, in addition to the N-terminal radical, of thearginine mimetic also enters into intermolecular interactions with thefactor X_(a) protein. Consequently, the factor X_(a) inhibitor accordingto the invention can be optimized both at the N-terminal radical and atthe C-terminal radical, which means that it is possible to provideinhibitor strengths which are markedly superior to those of the priorart.

A binding mode of this nature is surprising since all the argininemimetic-based factor X_(a) inhibitors which have thus far been disclosedin the prior art bind in a substrate-like manner. In the substrate-likebinding, the arginine mimetic binds in the S1 pocket while the radicalwhich is linked N-terminally to the arginine mimetic by way of apotential P2 radical, such as a Gly spacer or the like, extends into theS3 or S4 pocket, respectively. In the case of the factor X_(a) inhibitoraccording to the invention, the arginine mimetic likewise binds in theS1 pocket but, in contrast to the factor X_(a) inhibitors known from theprior art, the radical which is bound C-terminally to the argininemimetic, and not the radical which is bound N-terminally, extends intothe S3 or S4 pocket of the factor X_(a) protein, respectively.

Within the context of the present invention, the following terms havethe following meaning unless expressly specified otherwise:

A N-terminal radical of the arginine mimetic, or a radical which islinked N-terminally to the arginine mimetic, is a radical which isbonded to the arginine mimetic by way of the N^(α) atom of theN-terminal amino group of the arginine mimetic or by way of the group inthe arginine mimetic which corresponds to the amino group of theunmodified arginine.

Correspondingly, within the context of the present invention, aC-terminal radical of the arginine mimetic, or a radical which is linkedC-terminally to the arginine mimetic, is understood as being a radicalwhich is bonded to the arginine mimetic by way of the C-terminal C atomof the carboxyl group of the arginine mimetic or by way of the group inthe arginine mimetic which corresponds to the carboxyl group of theunmodified arginine.

Within the context of the present invention, intermolecular interactionsare all forms of van der Waals interactions, such as electrostaticinteractions between charged radicals in the inhibitor and oppositelycharged groups in the factor X_(a) protein, interactions between polargroups in the inhibitor and oppositely polarized groups in the factorX_(a) protein, and also hydrophobic interactions between nonpolar groupsin the inhibitor and in the factor X_(a) protein, and the like, and alsohydrogen bonds between the inhibitor and the factor X_(a) protein.

In connection with the present invention, an arginine mimetic isunderstood as being a compound which possesses the same functionalcharacteristics as arginine or functional characteristics which aresimilar to those of arginine, e.g. a side chain having a positive chargeat physiological pH, as is characteristic for the guanidinium group ofthe side chain of arginine. Thus, an arginine mimetic can be an aminoacid analog of arginine, i.e. a compound in which the N-terminal aminogroup, the C-terminal carboxyl group and/or the side chain of argininehas been chemically modified.

Amino acid analogs in which the side chain comprises a substituted orunsubstituted, saturated or unsaturated, carbocylic or heterocyclicradical can, in particular, be used as arginine mimetics in the presentinvention. While a ring of this nature is preferably a phenyl ring, itcan also be a pyridine ring or a piperidine ring, or another saturatedor unsaturated or aromatic, carbocyclic or heterocyclic group, with itbeing possible for the heteroatom(s) to be nitrogen, oxygen and/orsulfur.

Substituents of such a previously mentioned carbocyclic or heterocyclicradical are preferably basic substituents such as amidino, guanidino,amino, alkylamino, aminoalkyl, amide substituents and the like. It isfurthermore also advantageously possible to use polar substituents suchas halogens, e.g. chlorine, hydroxyl or alkoxy. The abovementionedcarbocyclic or heterocyclic radicals can be substituted once or morethan once by the abovementioned substituents, with combinations of theabovementioned substituents also being possible.

Furthermore, within the context of the present invention, the termarginine mimetics also encompasses modifications of the N-terminal aminogroup and of the C-terminal carbonyl group, with the proviso that thesemodifications exhibit the same, or essentially the same, spatialconfigurations as are typical for the unmodified arginine backbone. Anexample of such a modification is the reduction of the C-terminalcarbonyl group to a CH₂ group.

In the present invention, it is in principle also possible to use otherarginine mimetics which are known from the prior art, or which arederived therefrom, with the proviso that the arginine mimetic meets thesteric requirements for a P1 substrate of the factor X_(a) protein andthe C-terminal radical of the arginine mimetic can enter intointermolecular interactions with the S3/S4 pocket of the factor X_(a)protein.

In the present invention, the greatest preference is given to using, asthe arginine mimetic, a phenylalanine analog which is substituted by abasic radical on the aromatic ring. Most preferably, the basicsubstituent is an amidino group at the 3 position of the aromatic ring.

When an amino acid analog, such as the above-described phenylalanineanalog, is used as the arginine mimetic, the above-described,advantageous binding mode of the factor X_(a) inhibitor according to theinvention is achieved by the chirality at the C^(α) atom of the argininemimetic, or at a corresponding chiral center of a backbone-modifiedarginine mimetic, being R, such that the radical which is linkedC-terminally to the arginine mimetic extends into the S3/S4 pocket ofthe factor X_(a) protein and can there enter into intermolecularinteractions with the hydrophobic groups of the S3/S4 pocket.

When used as an arginine mimetic, a (R)-chiral amino acid analog has theadditional advantage that it is more stable than (S)-chiral amino acidanalogs and that the inhibitor can consequently remain active for alonger period in the body when used pharmacologically.

In a preferred embodiment of the present invention, the C-terminalradical of the arginine mimetic comprises a linker which is bondeddirectly to the arginine mimetic and also an optionally substitutedhydrophobic group which can enter into intermolecular interactions withthe hydrophobic S3/S4 pocket of the factor X_(a) protein.

The linker is preferably of a size which is suitable for bridging the S2pocket of the factor X_(a) protein, i.e. its spatial configuration ispreferably similar to that of the natural P2 substrate Gly.

The hydrophobic group of the C-terminal radical preferably exhibits aspatial configuration which enables the hydrophobic group to fitoptimally into the S3/S4 pocket of the factor X_(a) protein. Inaddition, it is advantageous if the hydrophobic group is substituted byone or more basic substituents which are configured such that it ispossible for interactions to take place with negatively charged ornegatively polarized groups of the factor X_(a) protein in theneighborhood of the S3/S4 pocket. Preferred basic substituents areamidino, guanidino, amino, alkylamino, aminoalkyl and amidesubstituents, and the like.

The present invention relates, in particular, to compounds in accordancewith the following structural formula I:

in which R¹ comprises a linker L¹, which is directly bonded to thephenylalanine analog, and a substituted or unsubstituted, saturated orunsaturated group R⁴; R² comprises a linker L², which is bonded directlyto the phenylalanine analog, and a substituted or unsubstituted,saturated or unsaturated group R⁷; and R³ is a basic substituent at the3 or 4 position of the aromatic ring of the phenylalanine analog and thearomatic ring is optionally substituted by at least one furthersubstituent R^(Y), where z=0 to 4.

The linker L¹ is used for linking the group R⁴ to the nitrogen atom ofthe phenylalanine analog of the formula I. In this connection, L¹ can beany group which enables such a linkage to take place. Preference isgiven to L¹ being a group which is chemically and enzymically stable inorder to prevent the compound of the formula I breaking down when beingused as a pharmaceutical composition.

The linker L¹ can simply be a bond. In that case, R⁴-L¹NH . . . is R⁴—NH. . . Preferably, the linker L¹ comprises a group R^(x) having a chainlength of from 1 to 10 atoms, preferably of from 1 to 5 atoms, such asC₁–C₁₀, in particular C₁–C₅-alkyl, C₁–C₁₀-, in particular C₁–C₅-alkenyl,C₁–C₁₀-, in particular C₁–C₅-alkynyl, with this group also being able tocontain heteroatoms, in particular O, S or N, in the chain, e.g.(O—CH₂—CH₂)_(n) in which n=1 to 3. Particularly preferably, the linkerL¹ comprises, in addition to said group R^(x) or without said groupR^(x), a linking group which is bonded to the nitrogen as phenylalanineanalog.

Particularly preferably, R¹ comprises a linker L¹ which is capable offorming hydrogen bonds. Linkers L¹ or linking groups which are capableof forming hydrogen bonds, or potential hydrogen acceptors or donors,which are additionally preferred because of their geometry, compriselinkers, such as —CO—, —CO—NH— or —COO—, which, together with the NHgroup of the phenylalanine analog, form an amide bond (in the case of—CO—), a urea bond (in the case of —CO—NH—) or a urethane bond (in thecase of —COO—) An N-terminal linkage of the group R⁴ by way of an—SO₂-linker is likewise possible.

Examples of preferred R¹ radicals are —CO—R⁴, —CO—NH—R⁴ or —COOR⁴, andcorresponding sulfur groups —CS—R⁴, —CS—NH—R⁴ or —COSR⁴. Particularlypreferably, R¹ is =COOR⁴. Even more preferably, R¹=—CO—NH—R⁴. Theabovementioned group R^(x) can be arranged between the linking group andthe radical R⁴. It has been found that urea derivatives (L¹=—CO—NH—)inhibit FX_(a) outstandingly well and are extremely stable chemicallyand enzymically, for which reason they are particularly suitable asinhibitors of FX_(a).

However, the linker L¹ can also be glycine (—CO—CH₂—NH—) or anothernatural or unnatural amine acid (—CO—CHR—NH—).

The group or radical R⁴ is preferably a hydrophobic radical. However, itcan also be a hydrophilic radical or a radical which possesses ahydrophobic group which carries one or more hydrophilic substituents. R⁴can, for example, be a saturated or unsaturated, substituted orunsubstituted, noncyclic alkyl radical; a saturated or unsaturated,substituted or unsubstituted carbocyclic radical; or a saturated orunsaturated, substituted or unsubstituted heterocyclic radical.

R⁴ is preferably a C₁₋₃₀-alkyl-, C₂₋₃₀-alkenyl-, C₂₋₃₀-alkynyl-,C₃₋₃₀-cycloalkyl-, C₅₋₃₀-aryl-, C₃₋₃₀-heteroaryl-, C₆–C₃₀-alkaryl- orC₄₋₃₀-alkheteroaryl radical, with these radicals being able to carry oneor more substituents. R⁴ preferably comprises at least 4 C atoms, morepreferably at least 6 C atoms and preferably up to 24 C atoms, morepreferably up to 18 C atoms. Suitable heteroatoms which the R⁴ radicalcan contain are, for example, O, N, S and P. The radical R⁴ canfurthermore have one or more substituents. R⁴ is preferably a noncyclicC₁- to C₅-alkyl radical which is substituted by at least one radical R⁶,with R⁶ being selected from C_(n)H_(2n+1), where n=1 to 10. R⁴ isparticularly preferably t-butyl. In another preferred embodiment, R⁴ issubstituted or unsubstituted phenyl, benzyl, fluorenyl, naphthyl,—C(CH₃)₂—C₆H₅ or adamantyl. Most preferably, R⁴=adamantyl.

Particularly preference is furthermore given to R⁴ radicals which arelarger (on the basis of the volume occupied) than is the phenyl radical.

According to the invention, the linker L² which is bonded directly tothe phenylalanine analog is preferably of a size which is suitable forbridging the S2 pocket of the factor X_(a) protein, i.e. its spatialconfiguration is preferably similar to that of the natural P2 substrateGly. In this connection, the linker of R² is preferably —OR⁵—, —NH—R⁵—,—NH—NH—R⁵— or —CH₂R⁵, where R⁵ is a substituted or unsubstituted,saturated or unsaturated, carbocyclic, heterocyclic or noncyclic alkylradical, or can be a group R^(x), as defined above. Particularlypreferably, the linker is L²=—NH—R⁵—. However, the linker L² can alsosimply be a bond.

It is advantageous for a factor X_(a) inhibitor according to theinvention having the structural formula I if a hydrophobic R⁵ radical isC-terminally linked to the phenylalanine derivative by way of an esteror amide bond, with R⁵ particularly preferably being a substituted orunsubstituted C₁- to C₅-alkyl radical. In this connection, R⁵ can havethe formula —(CH₂)_(m), in which m=1 to 3. Particularly preferably, R⁵is =—CH₂—CH₂—. Furthermore, R² comprises a saturated or unsaturatedgroup R⁷ which is unsubstituted or substituted by one or more radicalsR⁸ and which can be a noncyclic radical but is, in particular, acarbocyclic radical, such as a cyclic alkyl, alkylaryl, arylalkyl oraryl radical or a heterocyclic radical which contains at least oneheteroatom, such as oxygen, nitrogen and/or sulfur, with R⁸ preferablybeing a basic substituent and/or a substituent which functions as ahydrogen bond donor or acceptor, and/or a halogen.

R⁷ is preferably a C₁₋₃₀-alkyl, C₂₋₃₀-alkenyl, C₂₋₃₀-alkynyl,C₃₋₃₀-cycloalkyl, C₅₋₃₀-aryl, C₃₋₃₀-heteroaryl, C₆–C₃₀-alkaryl orC₄₋₃₀-alkheteroaryl radical, with these radicals being able to carry oneor more substituents. R⁷ preferably comprises at least 4 C atoms, morepreferably at least 6 C atoms and preferably up to 24 C atoms, morepreferably up to 18 C atoms. Suitable heteroatoms which the R⁷ radicalcan contain are, for example, O, N, S and P. The R⁷ radical canadditionally possess one or more substituents.

In particular, R can be a phenyl, piperidine, pyrrol, furan, thiophene,pyridine, naphthalene, anthracene or indole radical which isunsubstituted or substituted by one or more R⁸ radicals. Other aromaticradicals, including fused aromatic or heteroaromatic radicals, arelikewise conceivable.

R⁸ is preferably a radical which is a positively charged radical underphysiological conditions, e.g. a pH of approx. 6.5–7.5.

Particularly preferably, R⁸ is an amidino, guanidino, amino, ester,alkylamino, aminoalkyl, cyano, amide or hydroxyl radical, or the like.

Particularly advantageously for the use of the compounds according tothe invention having the structural formula I as factor X_(a)inhibitors, a radical —NH—CHR⁹—COO—(CH₂)_(m)R⁷, in which m=1 to 5, R⁷ isas defined above and R⁹ is a derivatized or nonderivatized side chain ofa natural amino acid, and which can readily be produced synthetically byesterifying a natural or unnatural amino acid, can be used as theradical R².

In the present invention, R³ is preferably an amidino, guanidino, amino,alkylamino, aminoalkyl or amide radical, or the like, particularlypreferably an amidino radical. The aromatic ring of the phenylalanineradical is substituted at the 3 or/and 4 position, preferably at the 3position, by the radical R³, e.g. an amidino radical.

Furthermore, the ring can also advantageously be additionallysubstituted by one or more substituents R^(Y), where z=0 to 4.Preference is given to polar substituents, such as halogen, e.g.fluorine, chlorine, bromine, iodine, hydroxyl or alkoxy and/or basicsubstituents.

R^(Y) can preferably, in each case independently at each occurrence, bea halogen, e.g. fluorine, chlorine, bromine, iodine, —OH, —NH₂, -formyl,-acetyl, —OMe (Me=methyl), —OEt (Et=ethyl), NHMe, —NHEt, SH, SEt, SMe,NMe₂, —CH₃, —CH₂OH, —CH₂—CH₃, —NH—OH, —COOH, —COOMe, CN, NO₂ or —CH₂CH₃.

The groups mentioned for the substituent R^(y) are also preferredsubstituents for the other substituted groups mentioned herein (e.g. inthe case of R⁴, R⁵ and R⁷), unless explicitly indicated otherwise.

Surprisingly, it has been found that it is advantageous for inhibitingfactor X_(a) with a compound according to the invention of thestructural formula (I) if the phenylalanine analog is (R) chiral,because the C-terminal radical is then able to enter into intermolecularinteractions with the S3/S4 pocket of the factor X_(a) protein.Preference is therefore given to the abovementioned compounds being inthe (R) conformation. However, the invention also encompasses thecompounds in the (S) conformation, and also mixtures of (R) and (S)enantiomers.

A very powerful inhibitory effect on factor X_(a) was achieved using thecompound according to the inventionN-1-adamantyloxycarbonyl-D-3-amidinophenylalanine-(2-phenyl)-1-ethylamide.

An even more powerful inhibitory effect on factor X_(a) was observed inthe case of the compound according to the inventionN-(1-adamantylaminocarbonyl)-D-3-amidinophenylalanine-(2-phenyl)-1-ethylamide.

The compound according to the invention can be present in free form oras a pharmaceutically acceptable salt, for example as a hydrochloride.

In that which follows, the present invention is illustrated usingrepresentative compounds according to the invention. FIGS. 2 to 4 listtheir inhibitory strengths toward factor X_(a) and, by comparison,toward uPA, thrombin and trypsin.

The preference of factor X_(a) for 3-amidinophenylalanine derivatives ascompared with 4-amidinophenylalanine derivatives is in agreement withprevious publications (Maduskuie et al., see above), although apreference for the 4-amidino group was observed in the case of theinverse binding of the bisbenzamidine compound 2 (B. Gabriel et al., seeabove). Interestingly, the 4-guanidinoderivative 26 in no way meets thesteric requirements for a P1 radical for this enzyme since a dramaticloss in inhibitory activity is observed as compared with compound 19.This also applies to the other enzymes investigated, i.e. uPA, thrombinand trypsin.

Comparison of the racemic compound 15, as a free acid at the C terminus,with the racemic compound 17, as a C-terminal amide derivative, does notshow any important differences, an observation which is in agreementwith the crystal structure of des-Gla-factor X_(a)-complexed DX-9065a,in which the free carboxylate group extends into the surrounding solvent(H. Brandstetter et al. (1996), J. Biol. Chem. 271, 29988–29992). In asimilar way, it was observed, in the prior art, that the inhibition offactor X_(a) is not affected when the compound 2 is present as the esterderivative instead of having a free carboxyl (Gabriel et al., seeabove). In view of these results, the significantly increased inhibitoryeffect of compound 11, i.e. the C-terminal methyl ester derivative, ascompared with that of compound 15, having the free C-terminal carboxylgroup, is extremely surprising. Evidently, the nature of the bonddiffers from that of the Daiichi inhibitor DX-9065a (Brandstetter etal., see above) or from that of compound 1 (M. Renatus et al. (1998), J.Med. Chem. 41, 5445 to 5456), in that the ester group is involved in anew type of interaction in the vicinity of the S1 binding site.

The advantageous effect, which is described in the previous paragraph,on the inhibitory action is augmented when, for example, use is made ofa C-terminal ester or a C-terminal amide derivative which comprises ahydrophobic group and a linker which is of a suitable size for bridgingthe S2 pocket of the factor X_(a) protein, i.e. its spatialconfiguration is similar to that of the natural P2 substrate Gly. Thisis illustrated by the compounds according to the invention 29 to 31 (seeFIG. 4). Thus, in the case of the strongest inhibitor 31, the group—NH—CH₂—CH₂— corresponds to the previously described linker in that itexhibits virtually the same spatial extent as does a Gly radical—NH—CH₂—CO—. The preferred hydrophobic group is a substituted orunsubstituted aryl or alkylaryl group, as previously described, in orderto achieve optimal intermolecular interactions with the hydrophobicS3/S4 pocket of the factor X_(a) protein, and consequently a powerfulinhibitory effect.

The effect of the chirality of the 3-amidinophenylalanine derivative onthe inhibition of factor X_(a) is depicted using as an example apreferred embodiment of the present invention, i.e. the racemic compound11. The K_(i) values of the L-(compound 27) and of the D-enantiomer(compound 28) are listed in Table 3. It is not possible to ascertain anyclear preference between the L-(compound 27) and D-enantiomer (compound28) by carrying out a crystal structure analysis of the known trypsin/11complex and by carrying out modeling studies for trypsin and factorX_(a) based on this crystal structure. This is confirmed by the K_(i)values of trypsin, since trypsin recognizes both enantiomers 27 and 28with almost identical affinity, and only with a slight preference forthe D-enantiomer. On the other hand, when inhibiting factor X_(a), theD-enantiomer 28, with a K_(i)=0.39 μM, surprisingly exhibits an activitywhich is about 10 times greater than that of the L-enantiomer 27.Whereas the affinity of this type of inhibitor for trypsin and thrombinis only marginally influenced by the chirality of the3-amidinophenylalanine radical, uPA is, on the other hand, only capableof recognizing the L-enantiomer. This is, therefore, the first report ofthe inhibitory strength for factor X_(a) being dependent on thechirality of the arginine mimetic employed and, in particular, the firstreport of a R-chiral arginine mimetic being an effective inhibitor offactor X_(a).

This preference of factor X_(a) for the (R) chirality of the argininemimetic is an essential feature of the present invention which it wasnot possible to expect on the basis of the known investigations. Thefact that the chirality at this position has an influence on selectivitywith regard to the inhibition of uPA, trypsin and thrombin, since uPA isselective for the (S) chirality whereas both trypsin and thrombinrecognize both isomers with comparable affinities, is also surprising.

Modeling studies carried out on the complex of factor X_(a) and compound11, and based on the known crystal structure of factor X_(a) (K.Padmanabhan et al. (1993), J. Mol. Biol. 232, 947–966), indicate thatthe bonding is of the following nature: the benzamidino group ofN-1-adamantyloxycarbonyl-D-3-amidinophenylalanine methyl ester (28) liesin the S1 pocket while the adamantyl group is located in a slight recesssurrounded by the side chains of Trp 215, Glu 217 and Phe 174 south ofthe substrate S3/S4 aryl binding site. In this type of binding, theC-terminal ester group points in the direction of the S3/S4 substratebinding pocket, with this being able to explain the preference for the(R) chirality. It can be presumed that a similar binding mechanism alsooperates in the case of the particularly preferred compounds accordingto the invention, 29 to 31.

A comparison of hydrophobic head groups which are N-terminally linked tothe 3-amidinophenylalanine, such as of the tert-butyl group (compound8), of the 9-fluorenylmethyl group (compound 10), of the 1-adamantylgroup (compound 11) and of the benzyl group (compound 4), clearly showsthat nonplanar and nonaromatic groups are most suitable. Thus, thecompound 11, having the 1-adamantyl group, leads to a submicromolarinhibition of factor X_(a) and, at the same time to remarkableselectivity vis-à-vis uPA, thrombin and trypsin.

While, with the exception of dramatic effects on the inhibition of uPA,the replacement of the N-terminal urethane group, as a potential waterbond acceptor in compound 11, with the related urea group (compound 12)evidently does not have any effect on the hydrogen bond network in thissegment of the protease/inhibitor complex, it leads to a desirablestability towards acids, for example stomach acids.

With regard to selectivity, the most marked effects are achieved bymeans of a free carboxyl group at the C terminus, which group evidentlyimpairs the interactions with uPA, thrombin and trypsin at their activesites in a specific manner. In a similar way, a 4-guanidino groupimpairs the inhibition not only of factor X_(a) but also of othertrypsin-like enzymes which have been investigated.

The compounds according to the invention are synthesized by means of aprocess which comprises the following steps:

-   -   a) adding R⁴—NCO, R⁴—NCS, X—CO—R⁴, X—SO₂—R⁴, X—CO—NH—R⁴ or        X—COOR⁴ to D- or L-phenylalanine which possesses the basic        substituent R³, or a precursor of R³, at the 3 or 4 position;    -   b) where appropriate converting the precursor of R³ into the        substituent R³;    -   c) where appropriate adding YR⁵ to the reaction product from        step b).

In this connection, X can be Cl or an active ester. In the same way, theabovementioned compounds which contain the R⁴ radical can be added, ifpossible, in the form of their respective acid anhydrides.

The N derivatives of the racemic 3- and 4-amidinophenylalanine areobtained from the respective 3- and 4-cyano compounds, followed by theirconversion into the related amidino derivatives, or by directderivatization of the amidinophenylalanine. Owing to side reactionswhich arise as a result of the unprotected amidino group, and owing todifficulties in purifying the hydrophilic amidino compounds, preferenceis given to reaction sequences in which the amidino function isgenerated in conclusion. For converting the cyanophenylalaninederivatives into the corresponding amidino compounds, the cyano groupcan be converted into the amidino radical by adding hydroxylaminehydrochloride and subsequently performing catalytic hydrogenation.However, other modifications of the two step reaction for synthesizingN-benzyloxycarbonylamidinophenylalanine piperidide, which have beenreported by Stüber et al. (Stüber et al. (1998), Peptide Res. 8, 78–85),are also possible.

When R²=OR⁵, the C derivatives of the racemic 3- and4-amidinophenylalanine can be obtained by adding the correspondingalcohol, where appropriate in the presence of acid or DCC(dicyclohexylcarbodiimide). When R²=NHR⁵, it is possible to use thecorresponding amine or a corresponding amino acid, where appropriate inthe presence of condensing reagents which are customarily used inpeptide synthesis. The examples of such condensing reagents are HOBT andTBTU. An important aspect of the present invention is the use of thecompounds according to the invention for producing a composition foranticoagulatory therapy. In connection with the present invention,anticoagulatory therapy is understood as being the treatment of vasculardiseases in order to avoid thrombotic vascular occlusions(antithrombotic therapy). Therapies of this nature comprise theprophylaxis and therapy of the venous thromboses and lung embolisms andthe antithrombotic therapy of arterial thromboses and embolisms,including coronary heart diseases such as angina pectoris or acutemyocardial infarction, cerebrovascular blood flow disturbances, such astransient ischaemic attacks and cerebral infarctions, and peripheralarterial occlusion diseases. In addition, the compounds according to theinvention can be used for hemorheologic therapy, i.e. for improving theflowability of the blood.

The compounds according to the invention of the structural formula I canalso be conceived as being suitable inhibitors of other serineproteases, in particular of human thrombin, plasma kallikrein andplasmin. In connection with such an inhibitory effect, the compoundsaccording to the invention can be used for preventing or treatingphysiological reactions, blood coagulation and inflammatory processeswhich are catalyzed by the abovementioned class of enzymes.

The present invention furthermore relates to a pharmaceuticalcomposition which, where appropriate, comprises a pharmaceuticallyacceptable excipient and at least one of the compounds according to theinvention. Preference is given to the pharmaceutical compositioncomprising a therapeutically effective quantity of the compoundsaccording to the invention. A “therapeutically effective quantity” isunderstood as being a quantity of the compounds according to theinvention having the structural formula I which, when administered onits own to a mammal, or administered to a mammal in combination with anadditional therapeutic agent, exhibits therapeutic activity and is, inparticular, active antithrombotically or as an antitumor agent.

Within the context of the present invention, “administration incombination” or “combination therapy” means that the compounds accordingto the invention of the formula I and one or more additional therapeuticcompositions are administered alongside each other to the mammal to betreated. When administration takes place in combination, each componentcan either be administered at the same time or consecutively atdifferent times in any sequence. Consequently, each component can beadministered separately but sufficiently close to each otherchronologically to ensure that they provide the desired therapeuticeffect. Other anticoagulants (or coagulation inhibiting agents) whichcan be used in combination with the compounds according to the inventioninclude warfarin and heparin and other factor X_(a) inhibitors whichhave been described in the prior art.

The administration of the compounds according to the invention incombination with such additional therapeutic compositions can afford anadvantage as compared with the respective use of the compounds andcompositions on their own by, for example, making it possible to uselower doses in each case, thereby minimizing any possible side effects.

The compounds according to the invention are suitable, in particular,for treatment or prophylactic use in association with diseases which areassociated with a pathological expression or overexpression of factorX_(a) and/or involve an increase in factor X_(a) proteolytic activitywhich can in turn be responsible for tumor growth-promoting andmetastasis-promoting fibrin depositions.

Thus, the compounds according to the invention are able to efficientlyinhibit and/or prevent the growth and/or spread of malignant tumors andthe metastasis of tumors. The invention therefore also relates to theuse of the compounds according to the invention for producing anantitumor agent. In this connection, the factor X_(a) inhibitorsaccording to the invention can, where appropriate, be formulatedtogether with suitable pharmaceutical auxiliary substances or carriersubstances for the purpose of producing drugs. It is furthermorepossible, where appropriate, to use the factor X_(a) inhibitors togetherwith other tumor agents or other active compounds or with other types oftreatment, for example in combination with irradiation or surgicalinterventions. Tumors which exhibit factor X_(a) activity, and which aresuitable for being treated with the compounds according to theinvention, are, in particular, lung, bladder, liver and ovariancarcinomas, and also malignant melanomas and neuroblastomas.

The compounds according to the invention already inhibit FXa at lowconcentrations. For example, the compound 31 which is presented hereininhibits with an inhibitor constant Ki=0.074 μM. Whereas the desired FXainhibition already takes place at such low concentrations, bloodcoagulation (according to the APPT test) is only affected atsubstantially higher concentrations of the compounds according to theinvention. As a result, the compounds according to the invention can beused selectively for inhibiting FXa without blood coagulation beingaffected at the same time. In this way, it is possible to use thecompounds according to the invention for controlling cancer (whichcontrol is connected with the inhibition of FXa) while being able toavoid side-effects, such as bleeding (which is connected to bloodcoagulation). This constitutes a fundamental advantage of the compoundsaccording to the invention as compared with other FXa inhibitors, suchas the known DX-9065a (Kakkar et al., J. Clinical Pathology—ClinicalMolecular Pathology Edition 48(5):M288–M290, 1995; Gouinthibault et al.,British Journal of Haematology 90(3): 669–680; Nakata et al., CancerLetters 122(1–2): 127–133, 1998; Yoshida et al., Fibrinolysis &Proteolysis, 11(3): 147–154, 1997; Barendszjanson et al., Tumor Biology,19(2): 104–112, 1998; Donnelly et al., Thrombosis & Haemostasis, 79(5):1041–1047, 1998; Fielding et al., Blood, 91(5): 1802–1809, 1998; Tanabeet al., Thrombosis Research, 96(2): 135–143, 1999).

The pharmaceutical composition can be administered to human and animalsin all known ways, for example topically, orally, rectally orparenterally, for example subcutaneously or intravenously. In addition,it can also be administered in the form of tablets, sugar-coatedtablets, capsules, pellets, suppositories, solutions or transdermalsystems, such as plasters.

The compounds according to the invention can also be used as standard orreference compounds, for example as a quality standard or control intests or assays which include the inhibition of factor X_(a). Thesecompounds can be provided in a commercial kit, for example for use inpharmaceutical research encompassing factor X_(a).

The compounds according to the invention can also be used in diagnosticassays which include factor X_(a).

The compounds according to the invention can be administered in oraldosage forms such as tablets, capsules, pills, powders, granules,elixirs, tinctures, suspensions, syrups and emulsions. They can also beadministered intravenously, intraperitoneally, subcutaneously orintramuscularly, in each case using dosage forms which are well known tothe skilled person. While they can be administered on their own,preference is given to administering them together with a pharmaceuticalexcipient which is selected on the basis of the chosen route ofadministration and customary pharmaceutical procedures.

The dose of the compounds according to the invention will naturallydepend on a variety of known factors, such as the pharmacodynamiccharacteristics of the particular composition and its nature and theroute of administration; and on the species, the age, the sex, thehealth, the medical condition and the weight of the recipient, and otherknown factors. A skilled person is able, without further instruction, todetermine the quantity of the compound according to the invention whichis effective for producing a composition for antithrombotic therapy.

In general, when being used to achieve the abovementioned effects, thedaily oral dose of the respective active constituents will be in therange of about 0.001 to 1 000 mg/kg of body weight, preferably of fromabout 0.01 to 100 mg/kg of body weight, per day and, most preferably,from about 1.0 to 20 mg/kg per day. For intravenous administration, thedoses which are most preferred are in a range from about 1 to about 10mg/kg/min during an infusion at a constant rate. The compounds accordingto the invention can be administered in a single daily dose orsubdivided into doses which are given 2, 3 or 4 times daily.

The compounds according to the invention can also be administered inintranasal form or administered transdermally.

The compounds according to the invention are typically suitably selectedin admixture with suitable pharmaceutical diluents, excipients orvehicles (which are jointly termed pharmaceutical vehicles in that whichfollows), with regard to the intended form of administration and inagreement with conventional pharmaceutical procedures.

Examples, in the case of oral administration in the form of a tablet orcapsule, the active compound component, in the form of the compoundaccording to the invention, can be combined with an oral, nontoxic,pharmaceutically acceptable, inert vehicle such as lactose, starch,sucrose, glucose, methyl cellulose, magnesium stearate, dicalciumphosphate, calcium sulfate, mannitol, sorbitol and the like. For oraladministration in liquid form, oral active compound components can becombined with any oral, nontoxic, pharmaceutically acceptable inertvehicles such as ethanol, glycerol, water and the like.

Furthermore, if necessary or desired, it is also possible to usesuitable binders, lubricants, disintegrants and dyes in thepharmaceutical composition. Suitable binders include starch, gelatin,natural sugars such as glucose or beta-lactose, natural and syntheticrubbers, carboxymethyl cellulose, polyethylene glycol, waxes and thelike. Lubricants which are used in these dose forms include sodiumoleate, sodium stearate, magnesium stearate, sodium benzoate, sodiumacetate, sodium chloride and the like. The disintegrants comprise, interalia, starch, methyl cellulose, agar, bentonite and the like.

The compounds according to the invention can also be administered in theform of liposomal transport systems. Liposomes can be formed from alarge number of phospholipids, such as cholesterol, stearylamine orphosphatidylcholins.

The compounds according to the invention can also be linked to solublepolymers acting as active compound vehicles. These polymers includepolyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamide phenol or polyethylene oxidepolylysine which is substituted by palmitoyl radicals. Furthermore, thecompounds according to the invention can be coupled to a number ofbiodegradable polymers which are useful for achieving controlled releaseof an active compound, for example to polyglycolic acid, polylacticacid, copolymers of polyglycolic acid and polylactic acid,polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters,polyacetals and the like.

Lipid dosage forms for oral administration can comprise dyes orflavorings for the purpose of increasing patient acceptance.

In general, water, a suitable oil, salt solutions, aqueous dextrose(glucose) and related sugar solutions, and glycols, such as propyleneglycol or polyethylene glycols, are suitable vehicles for parenteralsolutions. Solutions for parenteral administration preferably contain awater-soluble salt of the active constituent, suitable stabilizers and,if necessary, buffering substances. Antioxidants, such as sodiumdisulfite, sodium sulfite or ascorbic acid, either alone or incombination, are suitable stabilizers.

Other suitable pharmaceutical vehicles are described in “Remington'sPharmaceutical Sciences”, Mack Publishing Company, which is a standardreference work in this area.

The compounds according to the invention can also be employed as leadsubstances. They can, in particular, be used for developing or findingother effective factor X_(a) inhibitors, for example using appropriatealgorithms, which may, where appropriate, be computer-assisted. Whenemployed as lead substances, the compounds according to the inventioncan be used, in particular, for developing novel antithrombotic andantitumor agents.

The following examples serve to explain the invention withoutrestricting it in any way.

EXAMPLES

All the solvents and reagents which are used in the following exampleswere of the highest commercially available quality and, if required,were further purified and dried using standard methods. Analytical HPLCwas carried out on ET 125/4 Nucleosil 100/C₈ columns (Macherey-Nagel,Düren, Germany) using a linear gradient of MeCN/2% H₃PO₄ of 5:95 (A) to80:20 (B) in 12 minutes. ESI-MS spectra were recorded on a Perkin ElmerAPI 165 mass spectrometer (Perkin Elmer, Langen, Germany). TLC wascarried out on silica gel 60 plates using the following solvent systems:(A) CHCl₃/MeOH/AcOH, 40:10:2; (B) CHCl₃/MeOH/AcOH, 20:20:1; (C)AcOEt/n-BuOH/H₂O/AcOH, 10:6:2:2; (D) CHCl₃/MeOH/AcOH, 190:10:2; (E)CHCl₃/MeOH/NH₃, 20:20:9; (F) CHCl₃/MeOH/AcOH, 10:20:1; (G)n-hexane/AcOEt/AcOH, 49:49:2.

The synthesis, and inhibitor constants, of the compounds 1 and 4 aretaken from the prior art (B. Gabriel (1998), Doctoral Thesis, TechnischeUniversität München [Munich Technical University]).N,N′-Dibenzyloxycarbonyl-N″-trifylguanidine was synthesized inaccordance with Feichtinger et al. (J. Org. Chem. 63, 3804–3805, 1998).4-Nitrophenylalanine was obtained from Bachem (Heidelberg, Germany).D,L-3-Cyanophenylalanine and D,L-4-cyanophenylalanine were obtained fromSennchemicals (Dielsdorf, Switzerland), while Boc-D-3-cyanophenylalanineand Boc-L-3-cyanophenylalanine were obtained from Syntetech (Albany,Oreg., USA). The two latter compounds were N^(α)-deprotected in 95% TFA(trifluoroacetic acid).

Example 1 Synthesis of N-tert-butyloxycarbonyl-D,L-3-cyanophenylalanine(5)

(Boc)₂O (5.74 g; 26.29 mmol) in dioxane (5 ml) was added to a stirredsolution of D,L-(3-cyano)phenylalanine (5 g; 26.29 mmol) in dioxane (25ml) and 1 M NaOH (26.3 ml). After one hour, the solution was evaporatedand the residue was partitioned between AcOEt and 5% aqueous KHSO₄solution. The aqueous phase was extracted three times with AcOEt and thecombined organic phases were dried (over Na₂SO₄) and evaporated,resulting in a pale yellow oil, which crystallized at 4° C.

Yield: 6.9 g (91%); TLC (solvent system B): R_(f) 0.77; HPLC: t_(R) 8.2min; MS m/z 291.0 (M+H)⁺, calculated M_(r)=290.1.

Example 2 Synthesis ofN-tert-butyloxycarbonyl-D,L-3-hydroxyamidinophenylalanine (6)

A solution of compound 5 (1 g; 3.44 mmol), hydroxylamine hydrochloride(359 mg; 5.17 mmol) and KOH (483 mg; 8.6 mmol) in EtOH (50 ml) wasboiled under reflux overnight. Insoluble KCl was filtered off and thesolution was evaporated and the residue was dissolved in water (30 ml)and acidified to pH 2.5 with 1 M HCl. The solution was washed twice withAcOEt (20 ml) and the product was subsequently extracted five times withwater-saturated n-BuOH. The combined n-BuOH layers were evaporated.

Yield: 870 mg (78%) of white foam; TLC (solvent system C): R_(f) 0.62;HPLC: t_(R) 5.3 min; MS m/z=324.0 (M+H)⁺, calculated M_(r)=323.2.

Example 3 Synthesis ofN-tert-butyloxycarbonyl-D,L-3-amidinophenylalanine hydrochloride (7)

The compound 6 (870 mg; 2.69 mmol) was hydrogenated in water (50 ml)over 10% Pd/C at 50° C. for a period of 5 h. The catalyst was filteredoff and the solution was evaporated to dryness in the added presence of1 M HCl (2.7 ml).

Yield: 710 mg (77%); TLC (solvent system C): R_(f) 0.18; HPLC: t_(R) 5.4min; MS m/z=308.4 (M+H)⁺, calculated M_(r)=307.2.

Example 4 Synthesis ofN-tert-butyloxycarbonyl-D,L-3-amidinophenylalanine methyl esterhydrochloride (8)

A solution of 7 in MeOH (5 ml) was acidified down to pH 2 with 6 M HCland stirred at room temperature for 24 h. The solution was evaporateddown to dryness.

Yield: quantitative; TLC (solvent system C): R_(f) 0.41; HPLC: t_(r) 5.9min; MS m/z=322.4 (M+H)⁺, calculated M_(r)=321.2.

Example 5 Synthesis of D,L-3-amidinophenylalanine methyl esterdihydrochloride (9)

A solution of 8 (230 mg; 0.64 mmol) in 6 M HCl in dioxane (5 ml) wasstirred at room temperature. After 1 h, the solution was evaporated downto dryness.

Yield: quantitative; TLC (solvent system B): R_(f) 0.10; MS m/z=222.2(M+H)⁺, calculated M_(r)=221.1.

Example 6 Synthesis ofN-9-fluorenylmethyloxycarbonyl-D,L-3-amidinophenylalanine methyl esterhydrochloride (10)

Fmoc-Cl (9-fluorenylmethoxycarbonyl chloride, 44 mg; 0.17 mmol) and TEA(triethylamine, 24 μl; 0.17 mmol) were added to a solution of compound 9(50 mg; 0.17 mmol) in DMF (500 μl). After 30 min at room temperature,TEA (12 μl) was added in order to complete the reaction. After 3 h, thesolvent was evaporated and the residue was dissolved in water. Afteracidifying down to pH 3 with 1 M HCl, the product was collected bycentrifugation and precipitated once again from AcOEt/diisopropyl ether.

Yield: 60 mg (73%); TLC (solvent system A): R_(f) 0.58; HPLC: t_(R) 8.0min; MS: m/z=444.0 (M+H)⁺, calculated M_(r=443.2.)

Example 7 Synthesis ofN-1-adamantyloxycarbonyl-D,L-3-amidinophenylalanine methyl esterhydrochloride (11)

Compound 11 was prepared essentially as described for compound 10 usingAdoc-F (1-adamantyloxycarbonyl fluoride) and was reprecipitated fromAcOEt/diisopropyl ether.

Yield: 86%; TLC (solvent system A): R_(f) 0.55; HPLC: t_(R) 7.6 min; MS:m/z=400.4 (M+H)⁺, calculated M_(r)=399.2.

Example 8 Synthesis ofN-1-adamantylaminocarbonyl-D,L-3-amidinophenylalanine methyl esterhydrochloride (12)

Compound 9 (46 mg, 0.156 mmol) was reacted for 3 h in DMF (500 μl)containing 1-adamantyl isocyanate (27.7 mg; 0.156 mmol) and TEA (22 μl,0.156 mmol). After the solvent had been evaporated, the residue wascrystallized from isopropanol/diisopropyl ether.

Yield: 55 mg (81%); HPLC: t_(R) 8.7 min; MS m/z=399.4 (M+H)⁺, calculatedM_(r)=398.2.

Example 9 Synthesis of N-1-adamantyloxycarbonyl-D,L-3-cyanophenylalanine(13)

A solution of D,L-(3-cyano)phenylalanine (2 g; 10.5 mmol), Adoc-F (2.08g; 10.5 mmol) and 2 M NaOH (7.8 ml; 15.6 mmol) in dioxane (50 ml) wasstirred at room temperature for 3 h. The residue was partitioned betweenAcOEt and 5% aqueous KHSO₄ solution. The aqueous phase was extractedthree times with AcOEt and the combined organic phases were washed withsalt solution, dried (over Na₂SO₄) and evaporated. The resultingyellowish oil was treated with diethyl ether and evaporated down to awhite foam.

Yield: 3.7 g (96%); HPLC: t_(R) 8.7 min; TLC (solvent system B): R_(f)0.72; MS m/z=369.5 (M+H)⁺, calculated M_(r)=368.2.

Example 10 Synthesis ofN-1-adamantyloxycarbonyl-D,L-3-hydroxyamidinophenylalanine hydrochloride(14)

Compound 13 (3.7 g; 10 mmol) was reacted with hydroxylaminehydrochloride and worked up as described for compound 6.

Yield: 3.9 g (97%); HPLC: t_(R) 9.1 min; TLC (solvent system B): R_(f)0. 66; MS m/z=402.4 (M+H)⁺, calculated M_(r)=401.2.

Example 11 Synthesis ofN-1-adamantyloxylcarbonyl-D,L-3-amidinophenylalanine hydrochloride (15)

The catalytic reduction of compound 13 (3.9 g; 9.7 mmol) was carried outas described for compound 7.

Yield: 3.5 g (86%); HPLC: t_(R) 9.4 min; MS: m/z=386.4 (M+H)⁺,calculated M_(r)=385.2.

Example 12 Synthesis ofN-1-adamantyloxycarbonyl-D,L-3-cyanophenylalanine piperidide (16)

SOCl₂ (120 μl; 1.63 mmol) was added dropwise, at 0° C. and whilestirring vigorously, to a solution of compound 13 (300 mg; 0.814 mmol)and piperidine (480 μl; 4.88 mmol) in methylene chloride (5 ml). Afterthe mixture had been allowed to warm up to room temperature, and after 2h, the reaction mixture was diluted with methylene chloride and washedwith 5% aqueous NaHCO₃, 5% aqueous KHSO₄ solution, water and saltsolution, and dried (over Na₂SO₄). The solution was brought to dryness.

Yield: 240 mg (68%); TLC (solvent system D): R_(f) 0.76; MS m/z=436.2(M+H)⁺, calculated M_(r)=435.3.

Example 13 Synthesis ofN-1-adamantyloxycarbonyl-D,L-3-amidinophenylalanine piperididehydrochloride (17)

The reaction of compound 16 (240 mg; 0.55 mmol) with hydroxylaminehydrochloride, and the following catalytic reduction to give thecompound 17, were carried out as described for compounds 6 and 7.

Yield: 84 mg (31% over the two steps); HPLC: t_(R) 10.0 min; MS:m/z=453.4 (M+H)⁺, calculated M_(r)=452.3.

Example 14 Synthesis ofN-tert-butyloxycarbonyl-D,L-(4-amidino)phenylalanine hydrochloride (18)

Compound 18 was synthesized starting from D,L-(4-cyano)phenylalanine, asdescribed for the 3-substituted phenylalanine 7.

Yield: 57% (over 3 steps); HPLC: t_(R) 5.2 min; MS m/z=308.4 (M+H)⁺,calculated M_(r)=307.2.

Example 15 Synthesis of D,L-4-amidinophenylalanine dihydrochloride (19)

The deprotection of compound 18 (625 mg; 2.037 mmol) was carried out in6 M HCl in dioxane as described for compound 9.

Yield: 540 mg (95%); TLC (solvent system E): R_(f) 0.23; MS: m/z=208.3(M+H)⁺, calculated M_(r)=207.2.

Example 16 Synthesis of D,L-4-amidinophenylalanine methyl esterdihydrochloride (20)

SOCL₂ (180 μl; 2.47 mmol) was added dropwise, at −70° C. and whilestirring vigorously, to a solution of compound 17 (230 mg; 0.824 mmol)in MeOH (2 ml). The reaction mixture was allowed to warm to roomtemperature and was stirred for 18 h. The solvent was evaporated and theproduct was crystallized from EtOH/diethyl ether.

Yield: 182 mg (75%); TLC (solvent system E): R_(f) 0.54; MS: m/z=222.4(M+H)⁺, calculated M_(r)=221.1.

Example 17 Synthesis ofN-1-adamantyloxycarbonyl-D,L-4-amidinophenylalanine methyl esterhydrochloride (21)

Compound 21 was prepared from compound 20 using Adoc-F as described forcompound 10.

Yield: 32 mg (43%); HPLC: t_(R) 9.4 min; MS: m/z=400.4 (M+H)⁺,calculated M_(r)=399.2.

Example 18 Synthesis of D,L-4-nitrophenylalanine methyl esterhydrochloride (22)

SOCl₂ (1.27 μl; 18.88 mmol) was added dropwise to an ice-cool solutionof (4-nitro)phenylalanine (1 g; 4.72 mmol) in MeOH (5 ml). After 20 h,the solvent was evaporated and the weakly yellow solid was washed withether and dried.

Yield: 1.19 g (97%); TLC (solvent system F): R_(f) 0.70; MS: m/z=225.2(M+H)⁺, calculated M_(r)=224.1.

Example 19 Synthesis ofN-1-adamantyloxycarbonyl-D,L-4-nitrophenylalanine methyl ester (23)

Compound 23 was prepared from compound 22 using Adoc-F as described forcompound 10.

Yield: 725 mg (94%); HPLC: t_(R) 12.7 min; MS: m/z=403.4 (M+H)⁺,calculated M_(r)=402.2.

Example 20 Synthesis ofN-1-adamantyloxylcarbonyl-D,L-4-aminophenylalanine methyl ester (24)

Compound 23 (725 mg; 1.8 mmol) was hydrogenated for 2 h over Pd/C inMeOH (20 ml); the catalyst was subsequently filtered off and the solventwas evaporated. The crude product was chromatographed through silica gel(eluent: n-hexane/AcOEt/AcOH, 49:49:2).

Yield: 556 mg (83%); HPLC: t_(R) 14.2 min; TLC (solvent system G): R_(f)0.54; MS: m/z=373.4 (M+H)⁺, calculated M_(r)=372.4.

Example 21 Synthesis ofN-1-adamantyloxycarbonyl-D,L-4-(N^(ω),N^(ω)-dibenzyloxycarbonyl)guanidinophenylalaninemethyl ester (25)

A solution of compound 24 (57 mg; 0.153 mmol),N,N′-dibenzyloxycarbonyl-N″-trifylguanidine (70 mg; 0.153 mmol) and TEA(21 μl; 0.153 mmol) in methylene chloride (500 μl) was stirred at 50° C.for three days in a sealed reaction vessel equipped with a screwclosure. The solvent was evaporated and the crude product in AcOEt (10ml) was washed twice with 5% aqueous KHSO₄, water and salt solution. Theorganic phase was dried (over Na₂SO₄) and evaporated.

Yield: 96 mg (92%) of a colorless oil; HPLC: t_(R) 14.5 min; MS:m/z=683.4 (M+H)⁺, calculated M_(r)=682.3.

Example 22 Synthesis ofN-1-adamantyloxycarbonyl-D,L-4-guanidinophenylalanine methyl esterhydrochloride (26)

Compound 26 was obtained by catalytically hydrogenating compound 25 (96mg; 0.141 mmol) over Pd/C in MeOH (5 ml) which contained 1 M HCl (140μl; 0.141 mmol). The catalyst was filtered off, the solution wasevaporated and the residue was recrystallized from AcOEt/diisopropylether.

Yield: 53 mg (83%); HPLC: t_(R) 9.5 min; MS m/z=415.4 (M+H)⁺, calculatedM_(r)=414.2.

Example 23 Synthesis ofN-1-adamantyloxycarbonyl-L-3-amidinophenylalanine methyl esterhydrochloride (27)

Compound 27 was synthesized as described for compound 11, preceding fromL-(3-cyano)phenylalanine.

HPLC: t_(R) 9.6 min; MS: m/z=400.2 (M+H)⁺, calculated M_(r)=399.2.

Example 24 Synthesis ofN-1-adamantyloxycarbonyl-D-3-amidinophenylalanine methyl esterhydrochloride (28)

Compound 28 was synthesized as described for compound 11, proceedingfrom D-(3-cyano)phenylalanine.

HPLC: t_(R) 8.2 min; MS: m/z=400.4 (M+H)⁺, calculated M_(r)=399.2.

Example 25 Synthesis ofN-1-adamantyloxycarbonyl-D-3-amidinophenylalanine propylamidehydrochloride (29)

N^(a)-Adoc-D-3-Amidinophenylalanine hydrochloride (50 mg; 0.118 mmol),n-propylamine (29 μl; 0.354 mmol) and HOBT (19 mg; 0.142 mmol) weredissolved in 2 ml of DMF. TBTU (46 mg; 0.142 mmol) was added and thereaction mixture was stirred at room temperature for 3 h. After thesolvent had been evaporated off in vacuo, the resulting oil wasdissolved in 20 ml of ethyl acetate. The product began to precipitateout immediately and was separated off by centrifugation. The colorlesssolid was washed with ethyl acetate and dried in vacuo.

Yield: 26 mg (47%); HPLC: t_(R) 7.8 min; MS: m/z=427.4 (M+H)⁺,calculated M_(r)=426.3.

Example 26 Synthesis ofN-1-adamantyloxycarbonyl-D-3-amidinophenylalanine benzylamidehydrochloride (30)

N^(a)-Adoc-D-3-Amidinophenylalanine hydrochloride (30 mg; 0.071 mmol),benzylamine (23 μl; 0.213 mmol) and HOBT (11 mg; 0.085 mmol) weredissolved in 2 ml of DMF. TBTU (27 mg; 0.085 mmol) was added and thereaction mixture was stirred at room temperature. After 3 h, theprecipitated salt was filtered off and the solvent was evaporated invacuo. The residue was dissolved in 10 ml of ethyl acetate, after whichthis solution was washed with 5% aqueous NaHCO₃ solution and saltsolution and dried over anhydrous Na₂SO4. After the solvent had beenevaporated off in vacuo, the crude product was dissolved in 1 ml ofethyl acetate. 10 μl of 6N HCl in dioxane were added and the product wasprecipitated with tert-butyl methyl ether. The flocculent precipitatewas washed with diethyl ether and dried in vacuo.

Yield: 16 mg (43%); HPLC: t_(R) 10.3 min; MS: m/z=475.2 (M+H)⁺,calculated M_(r)=474.3.

Example 27 Synthesis ofN-1-adamantyloxycarbonyl-D-3-amidinophenylalanine-(2-phenyl)-1-ethylaminehydrochloride (31)

N^(a)-Adoc-D-3-Amidinophenylalanine hydrochloride (30 mg; 0.071 mmol),phenethylamine (27 μl; 0.213 mmol) and HOBT (11 mg; 0.085 mmol) weredissolved in 2 ml of DMF. TBTU (27 mg; 0.085 mmol) was added and thereaction mixture was stirred at room temperature. After 3 h, thereaction had not come to an end and 15 mg of TBTU (0.047 mmol) wereadded and the mixture was stirred for a further 3 h. The solvent wasevaporated in vacuo. The residue was dissolved in 10 ml of ethyl acetateand this solution was washed three times with 5% aqueous NaHCO₃ solutionand 1× with 2 ml of 0.5 N HCl and dried over anhydrous Na₂SO₄. After thesolvent had been evaporated off in vacuo, the product was precipitatedfrom iPrOH/DIPE. The flocculent precipitate was washed with diethylether and dried in vacuo.

Yield: 10 ml (27%); HPLC: t_(R) 6.8 min; MS m/z=489.4 (M+H)⁺, calculatedM_(r)=488.3.

Example 27a

Compounds 32 to 36 were synthesized in an analogous manner to theabove-described preparation methods.

Compound 32, containing the linker —NH—CO—NH—, inhibits FXa better, by afactor of 3, than does compound 31, containing the linker —O—CO—NH—.

Example 28 Determining the Inhibitor Constants

The measurements were carried out at 25° C. on a microplate reader (MR5000, Dynatech, Denkendorf, Germany). The test medium consisted of 200μl of Tris buffer (0.05 M; 0.154 M NaCl, 5% ethanol, pH 8.0), 25 μl ofaqueous substrate solution and 50 μl of enzyme solution. Twoconcentrations of the substrate and five concentrations of the inhibitorwere used. Three minutes after adding the enzyme, 25 μl of acetic acid(50%) were added in order to quench the reaction and the optical densitywas measured at 405 nm. The K_(i) values were calculated in accordancewith Dixon (M. Dixon (1953), Biochem. J. 55, 170–171) using a linearregression. The K_(i) values given in FIGS. 2 and 3 are means of atleast three determinations.

Example 29 Enzymes and Substrates for the K_(i) Determination

The following enzymes and the corresponding substrates were used at thegiven final concentrations: bovine thrombin, prepared in accordance withWalsmann (P. Walsmann (1968), Pharmazie 23, 401–402) (2 262 U/mg, finalconcentration 0.45 U/ml), substrate MeSO₂-D-hexahydrotyrosyl-Gly-Arg-pNA(final concentration 0.18 and 0.09 mM); bovine factor X_(a) (5 U/vial,0.11 U/ml; Diagnostic Reagents Ltd., Thame, UK), substrateMeSO₂-D-Nle-Gly-Arg-pNA (0.36 and 0.18 mM); human factor X_(a) (0.18μg/ml; Kordia Lab. Supplies, Leiden, Netherlands), substrate as forbovine factor X_(a) human plasmin (0.67 CTA U/mg, 0.06 CTA U/ml;Behringwerke AG, Marburg, Germany), substrate Tos-Gly-Pro-Lys-pNA (0.18and 0.09 mM); human uPA (500 000 U/vial, final concentration 150 U/ml;Ribosepharm GmbH Haan, Germany), substrate Bz-βAla-Gly-Arg-pNA (0.18 and0.09 mM); bovine pancreas trypsin (42 U/mg, 0.0038 U/ml; Serva,Heidelberg, Germany), substrate MeSO₂-D-hexahydrotyrosyl-Gly-Arg-pNA(0.18 and 0.06 mM).

The substrates were supplied by Pentapharm Ltd., Basel, Switzerland.

FIGURES

FIG. 1: Structures and inhibitor strengths of factor X_(a) inhibitors 1,2 and 3 from the prior art.

FIG. 2: Derivatives of 3-/4-amidino- or 4-guanidinophenylalanine,respectively, containing N^(α)-substituted carbamate or urea.

FIG. 3: Enantiomerically pure derivatives of1-adamantyloxycarbonyl-3-amidinophenylalanine methyl ester. K_(i) valuesmarked with * correspond to K_(i) values for human factor X_(a).

FIG. 4: Derivatives of N-1-adamantyloxycarbonyl-3-amidinophenylalaninecontaining C^(α)-substituted amide. K_(i) values marked with *correspond to K_(i) values for human factor X_(a).

FIG. 4 a: Other derivatives ofN-1-adamantyloxycarbonylamidinophenylalanine containingC^(α)-substituted amide, with compounds containing different R²radicals, and their K_(i) values, being depicted.

FIG. 5:N-(1-Adamantylaminocarbonyl)-D-3-amidino-phenylalanine-(2-phenyl)-1-ethylamideand its K_(i) values.

1. A compound of structural formula I:

wherein: L¹ is a linker which is a member selected from the groupconsisting of —CO—, —CO—NH—, and —COO; R⁴ is t-butyl, phenyl, benzyl,fluorenyl, or adamantyl; L² is a linker which is a member selected fromthe group consisting of a bond, —OR⁵—, —NH—R⁵—, —NH—NH—R⁵, and —CH₂—R⁵—,wherein R⁵ is —(CH₂)_(m)— and m is 1 to 3, or L² is a linker which isNH—CHR⁹—COO—(CH₂)_(m) wherein m is 1 to 5 and R⁹ is a derivatized orunderivatized side chain of a natural amino acid; R⁷ is a phenyl,piperidinepyrrol, furan, thiophene, pyridine, naphthalene, anthracene orindole radical which is unsubstituted or is substituted by one or moreR⁸ radicals, wherein R⁸ is a basic substituent or a substituentfunctioning as a hydrogen bond donor or acceptor or a halogen; and R³ isan amidino, guanidino, amino, alkylamino, aminoalkyl or amide radicaland is at the 3 position of the aromatic ring of the phenylalanineradical.
 2. The compound of claim 1, wherein R⁸ is an amidino,guanidino, amino, alkylamino, aminoalkyl, or amide radical.
 3. Thecompound of claim 1, wherein the phenylalanine radical is (R)-chiral. 4.N-1-Adamantyloxycarbonyl-D-3-amidophenylalanine-(2-phenyl)-1-ethylamide.5. N-1-Adamantyloxycarbonyl-D-3-amidophenylalanine-propylamide. 6.N-1-Adamantyloxycarbonyl-D-3-amidophenylalanine-propylamide benzylamide.7. The compound of claim 1, in the form of a pharmaceutically acceptablesalt.
 8. The compound of claim 7, wherein the pharmaceuticallyacceptable salt is a hydrochloride.
 9. A pharmaceutical compositioncomprising the compound of claim 1 and a pharmaceutically acceptablevehicle. 10.N-1-adamantyloxycarbonyl-D3-amidino-phenylalanine-2-phenyl-1 ethylamide.